Method for improving intestinal flora

ABSTRACT

A method for improving intestinal flora increases the number of beneficial bacteria such as lactic acid bacteria and Bifidobacterium to improve intestinal flora by using an enzyme. The method involves providing a protease, which is a polypeptide having an amino acid sequence shown in SEQ ID NO: 1 that can increase beneficial bacteria such as lactic acid bacteria and Bifidobacterium in intestines to exert an excellent effect of improving intestinal flora.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 15/999357, filed Aug. 17, 2018, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/JP2017/005986, filed Feb. 17, 2017, designating the U.S. andpublished as WO 2017/142080 A1 on Aug. 24, 2017, which claims thebenefit of Japanese Patent Application No. 2016-028942, filed Feb. 18,2016. All applications for which a foreign or a domestic priority isclaimed are identified in the Application Data Sheet filed herewith andare hereby incorporated by reference in their entirety under 37 C.F.R. §1.57.

REFERENCE TO THE ELECTRONIC SEQUENCE LISTING

The present application is being filed along with an Electronic SequenceListing as an ASCII text file via EFS-Web. The Electronic SequenceListing is provided as a file entitled LEX024002D1SEQLIST.txt, createdand last saved on Oct. 5, 2021, which is 4,091 bytes in size.

TECHNICAL FIELD

The present invention relates to an agent for improving intestinal florawhich can increase the number of beneficial bacteria such as lactic acidbacteria and Bifidobacterium to improve intestinal flora.

BACKGROUND

Recently, causal relationships between intestinal environment andvarious diseases are intensively investigated to reveal that improvementof intestinal environment is effective in preventing or amelioratingvarious diseases. In intestine, beneficial bacteria such as lactic acidbacteria and Bifidobacterium and bad bacteria such as Escherichia coliare present in a mixed manner Forming beneficial bacteria-dominatedflora is important in order to form a healthy intestinal environment.

Conventionally, probiotics, prebiotics, and the like are developed, andvarious materials by which beneficial bacteria become predominant toimprove intestinal flora are proposed. It is also reported thatadministration of digestive enzymes such as amylases and proteases canalso improve intestinal flora. For example, Non-Patent Document 1discloses that in a pig which is fed with a specific enzyme blendconsisting of an amylase, a protease, and a xylanase together withanimal feed, the number of Lactobacillus is increased and the number ofEscherichia coli is decreased in the large intestine. Non-PatentDocument 2 also discloses that in a pig which is fed with a specificenzyme blend (Nopcozyme II; Diasham Resources Pte Ltd.) consisting of anamylase derived from Bacillus amyloliquefaciens, a protease derived fromBacillus subtilis, and a xylanase derived from Trichoderma together withanimal feed, the number of Lactobacillus is increased and the numbers ofSalmonella and Escherichia coli are decreased.

Thus, as an enzyme preparation which can improve intestinal flora,specific enzyme blends consisting of an amylase, a protease, and axylanase are conventionally known. However, it has not been shown thatwhich enzyme of these enzymes contributes to the improvement ofintestinal flora. Further, there is a drawback that use of such enzymeblends leads to increases in cost of producing the enzymes.

REFERENCES

Non-Patent Document 1: Yi et al., Asian Astralas. J. Anim Sci., 2013,26: 1181-1188

Non-Patent Document 2: Zhang et al., J. Amin Sci., 2014, 92: 2063-2069

SUMMARY

Recently, in connection with increasing interest in health promotion,development of new materials by which beneficial bacteria becomepredominant to improve intestinal flora is desired. However, withrespect to an agent for improving intestinal flora using an enzyme, noeffective enzyme except for the conventionally reported enzymepreparations can be estimated even by analogy under the currentcircumstances.

Under the circumstances, an object of the present invention is toprovide an agent for improving intestinal flora which can increase thenumber of beneficial bacteria such as lactic acid bacteria andBifidobacterium to improve intestinal flora by using an enzyme.

The present inventors have made extensive investigations to solve theabove problem and found that a protease consisting of a polypeptidehaving an amino acid sequence shown in SEQ ID NO: 1 can increase thenumber of beneficial bacteria such as lactic acid bacteria andBifidobacterium in intestines to exert an excellent effect of improvingintestinal flora. The present inventors have made further investigationsbased on the findings, leading to the completion of the invention.

Thus, the present invention includes the following aspects.

Item 1. An agent for improving intestinal flora comprising a proteasecomprising at least one of the following polypeptides (1) to (3) as anactive ingredient: (1) a polypeptide consisting of an amino acidsequence shown in SEQ ID NO: 1, (2) a polypeptide comprising an aminoacid sequence in which one or a few amino acids are substituted, added,inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1,and having a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1, and (3) apolypeptide comprising an amino acid sequence having 80% or moresequence identity to an amino acid sequence shown in SEQ ID NO: 1, andhaving a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1.

Item 2. A drug for oral administration for improving intestinal flora,comprising an agent for improving intestinal flora according to Item 1.

Item 3. A food additive for improving intestinal flora, comprising anagent for improving intestinal flora according to Item 1.

Item 4. Food or drink for improving intestinal flora, comprising anagent for improving intestinal flora according to Item 1.

Item 5. A method for improving intestinal flora, comprising orallyapplying a protease comprising at least one of the followingpolypeptides (1) to (3) to a person who requires improvement ofintestinal flora: (1) a polypeptide consisting of an amino acid sequenceshown in SEQ ID NO: 1, (2) a polypeptide comprising an amino acidsequence in which one or a few amino acids are substituted, added,inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1,and having a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1, and (3) apolypeptide comprising an amino acid sequence having 80% or moresequence identity to an amino acid sequence shown in SEQ ID NO: 1, andhaving a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1.

Item 6. A protease for use in a treatment for improving intestinalflora, comprising at least one of the following polypeptides (1) to (3):(1) a polypeptide consisting of an amino acid sequence shown in SEQ IDNO: 1, (2) a polypeptide comprising an amino acid sequence in which oneor a few amino acids are substituted, added, inserted, or deleted in theamino acid sequence shown in SEQ ID NO: 1, and having a proteaseactivity equivalent to that of a polypeptide consisting of an amino acidsequence shown in SEQ ID NO: 1, and (3) a polypeptide comprising anamino acid sequence having 80% or more sequence identity to an aminoacid sequence shown in SEQ ID NO: 1, and having a protease activityequivalent to that of a polypeptide consisting of an amino acid sequenceshown in SEQ ID NO: 1.

Item 7. Use of a protease comprising at least one of the followingpolypeptides (1) to (3) for the manufacture of an agent for improvingintestinal flora: (1) a polypeptide consisting of an amino acid sequenceshown in SEQ ID NO: 1, (2) a polypeptide comprising an amino acidsequence in which one or a few amino acids are substituted, added,inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1,and having a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1, and (3) apolypeptide comprising an amino acid sequence having 80% or moresequence identity to an amino acid sequence shown in SEQ ID NO: 1, andhaving a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1.

The present invention can increase the number of beneficial bacteriasuch as lactic acid bacteria and Bifidobacterium and improve intestinalflora by using a specific protease, and thus is effective in forminghealthy intestinal environment, maintaining healthy intestinalenvironment, and preventing or treating a disease and/or symptom due inpart to unhealthy intestinal environment.

DETAILED DESCRIPTION

1. Agent for improving intestinal flora

An agent for improving intestinal flora of the present invention ischaracterized by containing a specific protease as an active ingredient.The agent for improving intestinal flora of the present invention isdescribed in detail below.

[Protease]

In the agent for improving intestinal flora of the present invention, aprotease comprising at least one of the following polypeptides (1) to(3) is used as an active ingredient. By selecting and using the specificprotease, beneficial bacteria such as lactic acid bacteria andBifidobacterium in intestines can be increased to improve intestinalflora effectively. (1) a polypeptide consisting of an amino acidsequence shown in SEQ ID NO: 1, (2) a polypeptide comprising an aminoacid sequence in which one or a few amino acids are substituted, added,inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1,and having a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1, and (3) apolypeptide comprising an amino acid sequence having 80% or moresequence identity to an amino acid sequence shown in SEQ ID NO: 1, andhaving a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1.

The polypeptide set forth in the above (1) is an acid protease derivedfrom Aspergillus oryzae, and polypeptides set forth in the above (2) and(3) are variants of the polypeptide set forth in the above (1).

In the polypeptide of the above (2), amino acid modifications introducedmay comprise any one of the modifications including substitution,addition, insertion, and deletion alone (e.g., substitution alone) orcomprise two or more of the modifications (e.g., substitution andinsertion). In the polypeptide of the above (2), the number of aminoacids which is substituted, added, inserted, or deleted may be one or afew, and examples include 1 to 81, preferably 1 to 48 or 1 to 32, morepreferably 1 to 16, 1 to 10, or 1 to 8, even more preferably 1 to 3, 1or 2, or 1.

In the polypeptide of the above (3), sequence identity to the amino acidsequence shown in SEQ ID NO: 1 may be 80% or more, and is preferably 85%or more, preferably 90% or more, more preferably 95% or more, even morepreferably 99% or more.

Herein, in the a polypeptide of the above (3), the sequence identity tothe amino acid sequence shown in SEQ ID NO: 1 refers to a sequenceidentity calculated by comparison with the amino acid sequence shown inSEQ ID NO: 1. The “sequence identity” refers to a value of amino acidsequence identity obtained by b12seq program (Tatiana A. Tatsusova,Thomas L. Madden, FEMS Microbiol.Lett., Vol.174, p247-250, 1999) inBLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available fromNational Center for Biotechnology Information (NCBI)]. Parametersettings may be as follows: Gap insertion Cost value: 11 and Gapextension Cost value: 1.

In polypeptides of the above (2) and (3), when an amino acidsubstitution is introduced in the amino acid sequence shown in SEQ IDNO: 1, examples of the amino acid substitution introduced include aconservative substitution according to a preferred aspect. That is,examples of the substitution in the polypeptides of the above (2) and(3) include the following substitutions: when an amino acid to besubstituted is a non-polar amino acid, a substitution with othernon-polar amino acids; when an amino acid to be substituted is anon-charged amino acid, a substitution with other non-charged aminoacids; when an amino acid to be substituted is an acidic amino acid, asubstitution with other acidic amino acids; and when an amino acid to besubstituted is a basic amino acid, a substitution with other basic aminoacids.

In the polypeptides of the above (2) and (3), the phrase “having aprotease activity equivalent to that of a polypeptide consisting of anamino acid sequence shown in SEQ ID NO: 1” refers to showing a proteaseactivity equivalent to that of the polypeptide of the above (1) when theprotease activities are measured under the following conditions (i.e.,showing a protease activity of about 80 to 120% as compared to theprotease activity (100%) of the polypeptide of the above (1)). (Methodfor measuring protease activity)

In a test tube, 5 mL of a 0.6 wt % casein solution (pH 3.0) is placedand maintained at 37° C. Then, 1 mL of an enzyme solution (in water as asolvent) containing a sample which is to be measured for a proteaseactivity is added and allowed to stand at 37° C. for exactly 10 minutes,and then 5 mL of a 0.44 mol/L trichloroacetic acid solution is added tostop the reaction. The mixture is allowed to stand at 37° C. for 30minutes followed by filtration with filter paper to obtain 2 mL offiltrate, which is transferred into another test tube, and then 5 mL of0.55 mol/L sodium carbonate and 1 mL of 3-fold diluted Folin's reagentare added thereto in this order. The mixture is allowed to stand at 37°C. for 30 minutes followed by measurement of absorbance at a wavelengthof 660 nm. In blank, the enzyme solution is added after the addition ofthe trichloroacetic acid solution. Separately, a standard curve fortyrosine is constructed using 10 to 40 μg/mL of tyrosine solutions bythe same procedure as the above-described procedure for filtrate. Underthe above conditions, an amount of an enzyme which causes an increase incolored materials by Folin's reagent corresponding to 1 μg of tyrosineper minute is defined as 1 U. The following equations are used for thecalculation.

[Equation 1]

Protease activity per 1 g or 1 mL of sample (U/g orU/mL)=(AT−AB)×F×(11/2)×(1/10)×1/W

AT: Absorbance at wavelength of 660 nm

AB: Absorbance at wavelength of 660 nm in blank

F: Amount of tyrosine (μg) corresponding to difference in absorbance of1 as determined by standard curve for tyrosine

11/2: Factor of conversion after completion of reaction into totalliquid volume

1/10: Factor of conversion into per minute of reaction

W: Amount of sample (g or mL) in 1 mL of enzyme solution

The polypeptide of the above (1) to (3) can be obtained by a method ofculturing Aspergillus oryzae which produces the polypeptide, and alsoobtained by a publicly-known genetic engineering technique.

[Dose of protease]

The agent for improving intestinal flora of the present invention may beapplied at a suitable dose as determined according to, for example,types of products in which the agent is used, applications, expectedeffects, and dosage forms. Examples of a daily dose for an adult humanas an amount of the above protease ingested or administered include 0.1to 3,000 mg, preferably 1 to 2000 mg, more preferably 1 to 1000 mg, evenmore preferably 2 to 500 mg, still more preferably 2 to 150 mg, mostpreferably 5 to 100 mg.

[Use of agent for improving intestinal flora]

The agent for improving intestinal flora of the present invention canincrease the number of beneficial bacteria such as Bifidobacterium andlactic acid bacteria in intestines by the effect of the above proteaseto form a beneficial bacteria-dominated flora, and thus is used for thepurpose of forming healthy intestinal environment, maintaining healthyintestinal environment, and the like. Specifically, the agent forimproving intestinal flora of the present invention is superior in aneffect of increasing the number of bacteria of Bifidobacterium spp. andLactobacillus spp. in intestines, and thus can also be used as an agentfor increasing the number of enteric bacteria of Bifidobacterium spp.and Lactobacillus spp. in intestines.

In addition, the agent for improving intestinal flora of the presentinvention can form a beneficial bacteria-dominated flora to form ahealthy intestinal environment, and thus can also be used for thepurpose of preventing or treating a disease and/or symptom due in partto unhealthy intestinal environment. Examples of the disease and/orsymptom include decreased immunity, colorectal cancer, allergic disease,nonalcoholic steatohepatitis, obesity, and inflammatory bowel disease.

[Form for using agent for improving intestinal flora]

The agent for improving intestinal flora of the present invention isorally applied by oral ingestion or oral administration. The agent forimproving intestinal flora of the present invention is orally ingestedor orally administered to exert an effect of improving intestinal floraafter arriving at intestines, and thus can be blended for use withvarious products such as food and drink, drugs for oral administration,animal feed, and pet food.

When the agent for improving intestinal flora of the present inventionis blended with the above various products, the products may containprobiotics and/or prebiotics as required together with the agent forimproving intestinal flora of the present invention.

Examples of a microorganism used as the probiotics include lactic acidbacteria, Bifidobacterium, and Bacillus subtilis var natto. Specificexamples of the lactic acid bacteria include lactic acid bacteria ofLactobacillus spp. such as Lactobacillus casei, Lactobacillusacidophilus, and Lactobacillus plantarum; lactic acid bacteria ofEnterococcus spp. such as Enterococcus faecalis, Enterococcus faecium,and Enterococcus hirae; and lactic acid bacteria of Streptococcus spp.such as Streptococcus bovis and Streptococcus thermophilus. Specificexamples of Bifidobacterium include Bifidobacterium adolescentis,Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacteriumpseudolongum, and Bifidobacterium thermophilum. These probiotics may beused alone or in combination of two or more.

Examples of the prebiotics include xylooligosaccharide,fructooligosaccharide, soybean oligosaccharides,isomaltooligosaccharide, lacto-fructo-oligosaccharides,galactooligosaccharides, and lactulose. These prebiotics may be usedalone or in combination of two or more.

A formulation of a product with which the agent for improving intestinalflora of the present invention is blended may be any one of solid form,semi-solid form, liquid form, and the like, and is suitably selectedaccording to types and applications of the product.

When the agent for improving intestinal flora of the present inventionis used for food and drink, the above protease is provided, as food anddrink for improving intestinal flora, solely or in combination withother food materials or additives to be prepared in a desired form.Examples of the food and drink include a food for specified health uses,a nutritional supplement, a functional food, and a food for patients inaddition to common food and drink. Forms of these foods and drinks arenot specifically limited. Specific examples include supplements such astablets, granules, powders, capsules, and soft capsules; and drinks suchas energy drinks, fruit drinks, carbonated beverages, and lactic aciddrinks.

When the agent for improving intestinal flora of the present inventionis used for food and drink, an amount of the agent blended into the foodand drink varies according to, for example, forms of the food and drink.In supplements, the agent may be blended, for example, so that an amountof the above protease is in a range of 0.03 to 1,000 mg/g, preferably0.3 to 700 mg/g, more preferably 0.3 to 350 mg/g, even more preferably0.6 to 170 mg/g, still more preferably 0.6 to 80 mg/g, most preferably1.5 to 50 mg/g. In drinks, the agent may be blended, for example, sothat an amount of the above protease is in a range of 0.0003 to 10mg/mL, preferably 0.003 to 7 mg/mL, more preferably 0.003 to 3.5 mg/mL,even more preferably 0.006 to 1.7 mg/mL, still more preferably 0.006 to0.8 mg/mL, most preferably 0.015 to 0.5 mg/mL.

When the agent for improving intestinal flora of the present inventionis used in the food and drink field, the agent for improving intestinalflora of the present invention can be provided, as a food additive,solely or in combination with other ingredients.

When the agent for improving intestinal flora of the present inventionis used for drugs for oral administration, the agent for improvingintestinal flora of the present invention is provided, as drugs for oraladministration which exert an effect of improving intestinal flora,solely or in combination with, for example, other pharmacologicallyactive ingredients, pharmaceutically acceptable bases, or additives tobe prepared in a desired form. Forms of the drugs are not specificallylimited. Specific examples include formulations for oral administrationsuch as tablets, granules, powders, capsules, soft capsules, syrups, andliquids.

Examples of the bases and the additives used for manufacturing the drugsfor oral administration include aqueous bases such as water and alcohol,oil-based bases, diluents, binders, filling agents, disintegrants,lubricants, algefacients, pH-adjusting agents, thickeners, antioxidants,sequestering agents, surfactants, emulsifiers, solubilizers,solubilizing agents, flavoring agents, and antiseptics.

When the agent for improving intestinal flora of the present inventionis used as drugs for oral administration, ratio of the agent blendedinto the drugs for oral administration can be suitably determinedaccording to, for example, forms of the drugs for oral administrationwithin a range satisfying the above described dose. In drugs for oraladministration in solid form or semi-solid form, the agent may beblended, for example, so that an amount of the above protease is in arange of 0.03 to 1,000 mg/g, preferably 0.3 to 700 mg/g, more preferably0.3 to 350 mg/g, even more preferably 0.6 to 170 mg/g, still morepreferably 0.6 to 80 mg/g, most preferably 1.5 to 50 mg/g. In drugs fororal administration in liquid form, the agent may be blended, forexample, so that an amount of the above protease is in a range of 0.0003to 10 mg/mL, preferably 0.003 to 7 mg/mL, more preferably 0.003 to 3.5mg/mL, even more preferably 0.006 to 1.7 mg/mL, still more preferably0.006 to 0.8 mg/mL, most preferably 0.015 to 0.5 mg/mL.

When the agent for improving intestinal flora of the present inventionis used for animal feed or pet food, the agent for improving intestinalflora of the present invention is provided, as animal feed or pet foodwhich exerts an effect of improving intestinal flora, solely or in adesired form controlled in combination with other animal feedingredients. Examples of the animal feed ingredients used for animalfeed or pet food include cereal crops such as corn, wheat, barley, andrye; brans such as wheat bran and rice bran; dregs such as corn glutenmeal and corn germ meal; animal-derived feed such as skimmed milkpowder, whey, fish flour, and powdered bone; yeasts such as brewer'syeast; calciums such as calcium phosphate and calcium carbonate;vitamins; amino acids; and saccharides.

When the agent for improving intestinal flora of the present inventionis used as animal feed or pet food, ratio of the agent blended into theanimal feed or pet food varies according to, for example, forms andtypes of the animal feed or pet food, and types of animals forapplication. For example, the agent may be blended so that an amount ofthe above protease is in a range of 0.00067 to 6.7 mg/g, preferably0.0067 to 6.7 mg/g, more preferably 0.0067 to 0.67 mg/g.

2. Other aspects

As described above, the above-described protease has an effect ofimproving intestinal flora. Thus, the present invention further providesa method for improving intestinal flora comprising orally applying theabove-described protease to a person who requires improvement ofintestinal flora, the above-described protease for use in a treatmentfor improving intestinal flora, and use of the above-described proteasefor the manufacture of an agent for improving intestinal flora. Theabove specific aspects of the present invention are as described in theabove section of “1. Agent for improving intestinal flora”.

EXAMPLE

The present invention is described more specifically below withreference to Example, but it should not be construed to be limited tothe example.

Test Example 1: Influence of polypeptide consisting of an amino acidsequence shown in SEQ ID NO: 1 on intestinal flora

1. Preparation of protease

A protease comprising a polypeptide consisting of an amino acid sequenceshown in SEQ ID NO: 1 was prepared from a koji extract obtained byculturing a microorganism which is a strain producing the protease by asolid-state fermentation. Specifically, Aspergillus oryzae was culturedon a solid culture medium comprising wheat bran at 25±5° C. for 3 days.The koji after the culturing was immersed in water to extract thepolypeptide, followed by removal of the koji by filtration throughdiatomaceous earth to obtain a polypeptide solution. Then, thepolypeptide solution was concentrated by using ultra filtrationmembrane, followed by desalting to obtain a partial purified polypeptidesolution. The partial purified polypeptide solution was sterilized byfiltration through a membrane filter, followed by spray drying using aspray dryer to give a power of a partially purified polypeptide. Thepartially purified polypeptide was subjected to a purification stepcomprising ion exchange column chromatography, hydrophobic columnchromatography, gel filtration chromatography, desalting columnchromatography, and freeze-drying in this order to yield a powder of apurified polypeptide. The resulting purified polypeptide was subjectedto SDS-polyacrylamide gel electrophoresis and CBB staining to detect aband of the polypeptide, which was treated with an enzyme followed byLC-MS/MS analysis and was confirmed to be a polypeptide consisting of anamino acid sequence shown in SEQ ID NO: 1.

2. Evaluation of effect of improving intestinal flora using rat

Using SD rats (male, 3 weeks old, from Hiroshima Institute forExperimental Animals), an influence of ingestion of a proteasecomprising a polypeptide having an amino acid sequence shown in SEQ IDNO: 1 on intestinal flora was investigated. In the experiment, the ratswere divided into 4 groups consisting of control groups 1 and 2 andworking groups 1 and 2, and the number of rats in each group was asfollows: control group 1:8 rats, control group 2:4 rats, working group1:4 rats, and working group 2:4 rats. The rats of each group were fed adlibitum for 14 days with animal feeds shown in Table 1.

TABLE 1 Control Working Working Control group 1 group 1 group 2 group 2Ingredient % w/w % w/w % w/w % w/w Beef tallow 30.00 30.00 30.00 30.00Casein 20.00 20.00 20.00 20.00 (Net protein: (Net protein: (Net protein:(Net protein: 17.40) 17.40) 17.40) 17.40) L-Cysteine 0.30 0.30 0.30 0.30Cellulose 5.00 5.00 5.00 5.00 Vitamin Mix ^(#1) 1.00 1.00 1.00 1.00Mineral Mix ^(#1) 3.50 3.50 3.50 3.50 Sucrose 20.00 20.00 20.00 20.00Corn starch 20.20 20.1904 20.1616 20.1616 Polypeptide ^(#2) — 0.00960.0384 — Inactivated polypeptide ^(#3) — — — 0.0384 ^(#1) refers to astandard purified diet for mice and rats according to AIN-93 (AmericanInstitute of Nutrition (AIN)). ^(#2) refers to a protease consisting ofa polypeptide of an amino acid sequence shown in SEQ ID NO: 1. ^(#3)refers to a polypeptide, which is a polypeptide of an amino acidsequence shown in SEQ ID NO: 1 inactivated under conditions of thetrichloroacetic acid treatment.

Bacteria in cecal contents of the SD rats were analyzed after 14-dayfeeding with the animal feed by real-time PCR using primers shown inTable 2 to calculate the numbers of Bifidobacterium (Bifidobacteriumspp.) and lactic acid bacteria (Lactobacillus spp.) in intestines.

TABLE 2 Base sequences of primers used For detectingForward: CGCGTCYGGTGTGAAAG (SEQ ID NO: 2) Bifidobacterium spp.Reverse: CCCCACATCCAGCATCCA (SEQ ID NO: 3) For detectingForward: GAGGCAGCAGTAGGGAATCTTC (SEQ Lactobacillus spp. ID NO: 4)Reverse: GGCCAGTTACTACCTCTATCCTTCTTC (SEQ ID NO: 5)

3. Results

The results are shown in Table 3. In the working groups 1 and 2, thenumbers of Bifidobacterium and lactic acid bacteria in intestines wereboth higher than those in the control groups 1 and 2. That is, it wasconfirmed that ingestion of the protease having the amino acid sequenceshown in SEQ ID NO: 1 promoted proliferations of Bifidobacterium andlactic acid bacteria, leading to improvement of intestinal flora. It wasalso confirmed that ingestion of a protease having an enzyme activity,which is not merely an administration of a peptide, improved intestinalflora.

TABLE 3 Control Working Working Control group 1 group 1 group 2 group 2Intestinal flora Bifidobacterium spp. 0.001 ± 0.000 0.282 ± 0.045 1.826± 0.214 0.005 ± 0.002 (% of total bacteria) Lactobacillus spp. 0.90 ±0.21 2.85 ± 0.68 3.05 ± 0.60 1.21 ± 0.53

What is claimed is:
 1. A method for improving intestinal flora,comprising orally applying a protease comprising at least one of thefollowing polypeptides (1) to (3) to a person who requires improvementof intestinal flora: (1) a polypeptide consisting of an amino acidsequence shown in SEQ ID NO: 1; (2) a polypeptide comprising an aminoacid sequence in which one or a few amino acids are substituted, added,inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1,and having a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1; and (3) apolypeptide comprising an amino acid sequence having 80% or moresequence identity to an amino acid sequence shown in SEQ ID NO: 1, andhaving a protease activity equivalent to that of a polypeptideconsisting of an amino acid sequence shown in SEQ ID NO: 1, wherein theprotease increase the number of Bifidobacteria and Lactic acid bacteriain intestines of said subject, thereby improves the intestinal flora. 2.The method of claim 1, wherein the oral application of the protease isperformed by orally applying an enzyme consisting of at least one of thepolypeptides (1) to (3).